A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

12A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a 13 division machinery in a genetic programme. A key event in the bacterial cell cycle is the assembly of 14 cytoskeletal proteins into a ring that defines the division site. At the onset of the formation of the 15 Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane recruiting proteins takes 16 place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be 17 reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures 18 are found to constrict the membrane and generate budding vesicles, a phenotype that has not been 19 reported before. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ 20 bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These 21 results provide new insights on the self-organization of basic cytoskeletal elements involved in bacterial 22 division. Moreover, they demonstrate that gene-directed protein synthesis and assembly of membrane-23 constricting FtsZ-rings can be combined in a liposome-based artificial cell. 24